Spontaneous 46,XX primary ovarian insufficiency (POI) affects as many as 1 in 100 women by the age of 40. The most common genetic cause identified for 46,XX POI is heterozygosity for the Fragile X (FX) premutation, In FXPOI, ovarian insufficiency results from increased follicular atresia in the ovaries of FMR1 premutation (CGG lengths from 60-200 repeats) carriers; however, the mechanism(s) that result in this atresia remain unclear. POI encompasses the spectrum of ovarian dysfunction from early indicators of ovarian failure, such as infertility and elevated levels of follicle-stimulating hormone, to featurs of overt ovarian failure, including irregular menstrual cycles. Understanding the role of the FMR1 premutation allele in ovarian dysfunction is critical to improving current assisted reproductive technologies and developing new therapeutic strategies. In this application, we propose to develop mouse models that will allow studies of the rCGG's role in FXPOI. Determining whether ectopically expressed premutation-length CGG repeat tracts are sufficient to confer POI is critical to understanding the role of rCGG in the pathogenesis of FXPOI. We propose to generate and study new mouse models designed to generate FXPOI pathology and capable of determining the role(s) of rCGG, FMR1 as well as tissue specificity of expression. Two aims are proposed: In Aim 1, we will create four new FXPOI mouse models. Two experimental models will contain a human genomic DNA fragment with a 90 repeat CGG tract upstream of a heterologous ZsGreen1 transcript (CGG90- ZsGreen1) or a human FMR1 cDNA. The targeting vectors will also include a loxP-flanked transcriptional/ translational STOP sequence (LSL) to allow for spatial and/or temporal control of expression of CGG repeat RNA using tissue-specific or drug-dependent Cre excision. Two control models will be created without the 90 repeat CGG to determine the specificity of rCGG to phenotypes. These models will allow us to assess the requirement of the FMR1 transcript in mediating pathology, and enable us to determine whether specific phenotypes associated with FXPOI originate from expression of rCGG in specific tissues of the hypothalamic- pituitary-gonadal axis and to compare these with those resulting from widespread expression. In Aim 2, we propose to study mice expressing the introduced constructs for features of FXPOI. We will compare our models for phenotypes observed in existing models that broadly express FMR1-coupled rCGG transgenes. We propose to count and determine quality of follicles in time course studies and to measure a variety of hormone levels for alterations from normal levels. We will also measure FMR1 mRNA and protein. We anticipate that these models will be of significant interest to the research community for additional studies of FXPOI, FXTAS and other phenotypes that result from rCGG repeat, and expect to distribute them widely.